Chimpanzee adenoviral vector-based filovirus vaccines

ABSTRACT

This invention provides vaccines for inducing an immune response and protection against filovirus infection for use as a preventative vaccine in humans. In particular, the invention provides chimpanzee adenoviral vectors expressing filovirus proteins from different strains of Ebolavirus (EBOV) or Marburg virus (MARV).

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a Divisonal Application of U.S. application Ser. No.13/641,655, filed Jan. 2, 2013, which is a U.S. National PhaseApplication of PCT/US2011/032682, filed Apr. 15, 2011 which claims thebenefit of U.S. Provisional Patent Application No. 61/325,166, filedApr. 16, 2010 each of which are incorporated herein by reference intheir entirety.

REFERENCE TO SEQUENCE LISTING

This application includes a Sequence Listing as a text file named“77867-591100US-854933 SEQLIST.txt” created Oct. 15, 2012, andcontaining 387,507 bytes. The material contained in this text file isincorporated by reference in its entirety for all purposes.

FIELD OF THE INVENTION

This invention relates generally to viral vaccines and, moreparticularly, to filovirus vaccines based on chimpanzee adenoviralvectors.

BACKGROUND OF THE INVENTION

The Ebola viruses, and the genetically-related Marburg virus, viruses ofthe Filoviridae family, are associated with outbreaks of highly lethalhemorrhagic fever in humans and primates in North America, Europe, andAfrica (Peters, C. J. et al. in: Fields Virology, eds. Fields, B. N. etal. 1161-1176, Philadelphia, Lippincott-Raven, 1996; Peters, C. J. etal. 1994 Semin Virol 5:147-154). Ebola viruses are negative-stranded RNAviruses comprised of five subtypes, including those described in theZaire, Sudan, Reston, Ivory Coast and Bundibugyo episodes (Sanchez, A.et al. 1996 PNAS USA 93:3602-3607). The Ebola virus, was firstrecognized during an outbreak in 1976 in the Ebola River valley of Zaire(currently the Democratic Republic of the Congo), Africa. Mortalityrates vary between different species, spanning from approximately 35 to90% for the most virulent ones, Zaire and Sudan. The development ofeffective vaccines and/or drugs is a high priority. The Ebola (EBOV) andMarburg (MARV) viruses have also been categorized as priority class Apathogens due to their virulence, ease of dissemination, lack ofeffective countermeasures to prevent or treat them, and their potentialto cause public panic and social disruption.

Although several subtypes have been defined, the genetic organization ofEbola viruses is similar, each containing seven linearly arrayed genes.Among the viral proteins, the envelope glycoprotein exists in twoalternative forms, a 50-70 kilodalton (kDa) secreted protein of unknownfunction encoded by the viral genome and a 130 kDa transmembraneglycoprotein generated by RNA editing that mediates viral entry (Peters,C. J. et al. in: Fields Virology, eds. Fields, B. N. et al. 1161-1176,Philadelphia, Lippincott-Raven, 1996; Sanchez, A. et al. 1996 PNAS USA93:3602-3607). Other structural gene products include the nucleoprotein(NP), matrix proteins VP24 and VP40, presumed nonstructural proteinsVP30 and VP35, and the viral polymerase (reviewed in Peters, C. J. etal. in: Fields Virology, eds. Fields, B. N. et al. 1161-1176,Philadelphia, Lippincott-Raven, 1996). Although spontaneous variation ofits RNA sequence does occur in nature, there appears to be lessnucleotide polymorphism within Ebola subtypes than among other RNAviruses (Sanchez, A. et al. 1996 PNAS USA 93:3602-3607), suggesting thatimmunization may be useful in protecting against this disease. Previousattempts to elicit protective immune responses against Ebola virus usingtraditional active and passive immunization approaches have, however,not succeeded in primates (Peters, C. J. et al. in: Fields Virology,eds. Fields, B. N. et al. 1161-1176, Philadelphia, Lippincott-Raven,1996; Clegg, J.C.S. et al. 1997 New Generation Vaccines, eds.: Levine,M. M. et al. 749-765, New York, N.Y. Marcel Dekker, Inc.; Jahrling, P.B. et al. 1996 Arch Virol Suppl 11:135-140).

Replication-defective adenovirus vectors (rAd) are powerful inducers ofcellular immune responses and have therefore come to serve as usefulvectors for gene-based vaccines, particularly for lentiviruses andfiloviruses, as well as other nonviral pathogens (Shiver, et al., (2002)Nature 415(6869): 331-5; (Hill, et al., Hum Vaccin 6(1): 78-83.;Sullivan, et al., (2000) Nature 408(6812): 605-9; Sullivan et al.,(2003) Nature 424(6949): 681-4; Sullivan, et al., (2006) PLoS Med 3(6):e177; Radosevic, et al., (2007); Santra, et al., (2009) Vaccine 27(42):5837-45. Adenovirus-based vaccines have several advantages as humanvaccines since they can be produced to high titers under GMP conditionsand have proven to be safe and immunogenic in humans (Asmuth, et al., JInfect Dis 201(1): 132-41; Kibuuka, et al., J Infect Dis 201(4): 600-7;Koup, et al., PLoS One 5(2): e9015.; Catanzaro, et al., (2006) J InfectDis 194(12): 1638-49; Harro, et al., (2009) Clin Vaccine Immunol 16(9):1285-92). While most of the initial vaccine work was conducted usingrAd5 due to its significant potency in eliciting broad antibody and CD8+T cell responses, pre-existing immunity to rAd5 in humans may limitefficacy (Catanzaro, (2006); Cheng, et al., (2007) PLoS Pathog 3(2):e25.; McCoy, et al., (2007) J Virol 81(12): 6594-604.; Buchbinder, etal., (2008) Lancet 372(9653): 1881-93). This property might restrict theuse of rAd5 in clinical applications for many vaccines that arecurrently in development including Ebola virus (EBOV) and Marburg virus(MARV).

To circumvent the issue of pre-existing immunity to rAd5, severalalternative vectors are currently under investigation. These includeadenoviral vectors derived from rare human serotypes and vectors derivedfrom other animals such as chimpanzees (Vogels, et al., (2003) J Virol77(15): 8263-71; Abbink, et al., (2007) J Virol 81: 4654-63; Santra,(2009) Vaccine 27(42): 5837-45). Chimpanzee adenoviral vectors are alsodescribed in WO 2010/086189, WO 2005/071093 and WO 98/10087.

It would thus be desirable to provide a vaccine to elicit an immuneresponse against a filovirus or disease caused by infection withfilovirus using improved adenoviral vectors. It would further bedesirable to provide methods of making and using said vaccine. Thepresent invention addresses these and other needs.

BRIEF SUMMARY OF THE INVENTION

This invention provides vaccines for inducing an immune response andprotection against filovirus infection for use as a preventative vaccinein humans. In particular, the invention provides chimpanzee adenoviralvectors (adenoviral vectors derived from chimpanzees) expressingfilovirus proteins. For example, these vaccines include chimpanzeeadenovirus serotypes ChAd3, ChAd63, PanAd3, PanAd1, PanAd2, or ChAd83expressing filovirus envelope glycoprotein (GP), including differentstrains of Ebolavirus (EBOV) or Marburg (MARV). Exemplary ChimpAdenoviral Ebola and Marburg sequences are provided in SEQ ID NOs:1-9.

DEFINITIONS

An “adenovirus capsid protein” refers to a protein on the capsid of anadenovirus (e.g., chimpanzee adenovirus) that is involved in determiningthe serotype and/or tropism of a particular adenovirus. Adenoviralcapsid proteins typically include the fiber, penton and/or hexonproteins. As used herein an “adenovirus capsid protein” may be, forexample, a chimeric capsid protein that includes capsid proteinsequences from two adenoviral iolates.

The terms “adjuvant” and “immune stimulant” are used interchangeablyherein, and are defined as one or more substances that cause stimulationof the immune system. In this context, an adjuvant is used to enhance animmune response to the adenovirus vectors of the invention.

The term “corresponding to”, when applied to positions of amino acidresidues in sequences, means corresponding positions in a plurality ofsequences when the sequences are optimally aligned.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, (e.g., adenovirus capsidproteins of the invention and polynucleotides that encode them) refer totwo or more sequences or subsequences that are the same or have aspecified percentage of amino acid residues or nucleotides that are thesame, when compared and aligned for maximum correspondence, as measuredusing one of the following sequence comparison algorithms or by visualinspection. Percentage of sequence identity is determined by comparingtwo optimally aligned sequences over a comparison window, wherein theportion of the polynucleotide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity.

An “isolated” nucleic acid molecule or adenovirus vector is a nucleicacid molecule (e.g., DNA or RNA) or virus, which has been removed fromits native environment. For example, recombinant DNA molecules containedin a vector are considered isolated for the purposes of the presentinvention. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe DNA molecules of the present invention. Isolated nucleic acidmolecules according to the present invention further include suchmolecules produced synthetically.

“Operably linked” indicates that two or more DNA segments are joinedtogether such that they function in concert for their intended purposes.For example, coding sequences are operably linked to promoter in thecorrect reading frame such that transcription initiates in the promoterand proceeds through the coding segment(s) to the terminator.

A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases typically read from the 5′to the 3′ end. Polynucleotides include RNA and DNA, and may be isolatedfrom natural sources, synthesized in vitro, or prepared from acombination of natural and synthetic molecules. When the term is appliedto double-stranded molecules it is used to denote overall length andwill be understood to be equivalent to the term “base pairs”.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 50 amino acid residues are commonly referred to as“oligopeptides”.

The term “promoter” is used herein for its art-recognized meaning todenote a portion of a gene containing DNA sequences that provide for thebinding of RNA polymerase and initiation of transcription of an operablylinked coding sequence. Promoter sequences are typically found in the 5′non-coding regions of genes.

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

The phrase “substantially identical,” in the context of two nucleicacids or polypeptides of the invention (e.g., adenovirus capsid proteinsor filovirus antigens), refers to two or more sequences or subsequencesthat have at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, and 95% nucleotideor amino acid residue identity, when compared and aligned for maximumcorrespondence, as measured using one of the following sequencecomparison algorithms or by visual inspection. In some embodiments, thesubstantial identity exists over a region of the sequences that is atleast about 50 residues, at least about 100 residues, or at least about150 residues in length. In one embodiment, the sequences aresubstantially identical over the entire length of the coding regions.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generally,Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).

Examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1990)J Mol. Biol.215: 403-410 and Altschuel et al. (1977) Nucleic Acids Res. 25:3389-3402, respectively. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation. This algorithm involves first identifying high scoringsequence pairs (HSPs) by identifying short words of length W in thequery sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al, supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, M=5, N=−4, and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915(1989)).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, less than about 0.01, or less thanabout 0.001.

A further indication that two nucleic acid sequences or polypeptides ofthe invention are substantially identical is that the polypeptideencoded by the first nucleic acid is immunologically cross reactive withthe polypeptide encoded by the second nucleic acid, as described below.Thus, a polypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions. Another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions, as described below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B Transgene expression by rAd5, ChAd63 and ChAd3 vectors. FIG.1A is a schematic of the genomic features of rAd vector. FIG. 1B showsEbola GP expression in HEK 293 cells. The cells were transduced withrAd5, ChAd63 or ChAd3 vectors at 0, or 10¹ to 10³ vp/cell as indicated.The cell lysates were harvested at 20 hours post transduction andsubjected to SDS-PAGE and Western blot analysis.

FIGS. 2A-2C ChAd3 Ebola GP (Zaire) single immunization generatedcomparable CD4+ T cell and IgG responses to rAd5. The mice wereimmunized with rAd5 Ebola (Zaire) or ChAd3 Ebola (Zaire) at 10⁷, 10⁸ and10⁹ vp intramuscularly. The spleens and sera were harvested 3 weeks postimmunization to detect cellular immune responses by ICS (intracellularcytokine staining) and IgG by ELISA. FIGS. 2A and 2B show %IFN-γ-producing CD4+ and CD8+ T cells, respectively. FIG. 2C showsdetection of IgG by ELISA Serum IgG (sera were diluted at 1:1000). *p<0.05; *** p<0.001.

FIGS. 3A-3C ChAd3 Ebola (Zaire) single immunization generated strongercellular and humoral responses than ChAd63. Mice were immunized withrAd5, ChAd3 or ChAd63 at 10⁹ vp intramuscularly. Spleens and serum wereharvested 3 weeks post immunization to detect cellular immune responsesby ICS and IgG response by ELISA. FIGS. 3A and 3B show % IFN-γ-producingCD4+ and CD8+ T cells, FIG. 3C shows Serum IgG (serum was diluted at1:1000). * p<0.05; *** p<0.001.

FIGS. 4A-4C ChAd3 Ebola (S/G) single immunization generated comparablecellular and humoral responses to rAd5. Mice were immunized with rAd5Ebola (S/G) or ChAd3 Ebola (S/G) at 107, 108 and 109 vp intramuscularly.Spleens and sera were harvested 3 weeks post immunization to detectcellular immune responses by ICS and IgG by ELISA. FIGS. 4A and 4B show% IFN-γ-producing CD4+ and CD8+ T cells, respectively. FIG. 4C showsSerum IgG (sera were diluted at 1:1000). * p<0.05; ** p<0.01.

FIG. 5. ChAd3 Ebola (Zaire) single immunization generatesantigen-specific antibody responses. Cynomolgus macaques were immunizedwith rAd5 or ChAd3 encoding EBOV-GP at a dose of 1011 vpintramuscularly. Serum was collected 4 weeks post immunization to detectIgG response by ELISA against EBOV GP.

FIGS. 6A-6B ChAd3 Ebola GP (Zaire) single immunization generatesantigen-specific CD4+ and CD8+ T cell responses. Cynomolgus macaqueswere immunized with rAd5 or ChAd3 encoding EBOV-GP at a dose of 10¹¹ vpintramuscularly. Blood cells were collected 4 weeks post immunization todetect cellular immune responses by intracellular cytokine stainingafter stimulation with EBOV-GP peptides. FIG. 6A shows %cytokine-producing CD4+ T cells, FIG. 6B shows % cytokine-producing CD8+T cells.

FIG. 7. ChAd3 Ebola GP (Zaire) single immunization protects nonhumanprimates against infectious challenge with a lethal dose of EBOV-Zaire.Cynomolgus macaques were immunized with rAd5 or ChAd3 encoding EBOV-GPat a dose of 10¹¹ vp intramuscularly. Subjects were challenged with 1000PFU of EBOV-Zaire by the intramuscular route at 5 weeks aftervaccination. *Additional 10 historical controls performed with the samevaccine and infectious virus challenge stock have yielded the samesurvival result, **More than 50 historical controls with the sameinfectious challenge stock have yielded the same survival result.

FIG. 8. A single immunization with 10¹⁰ vp of rAdC3 Ebola (Zaire)elicits antigen-specific Antibody responses. Cynomolgus macaques werevaccinated with rAdC3 or rAd5 encoding EBOV-GP at a dose of 10¹⁰ vpintramuscularly. Serum was collected at 4 weeks post Immunization todetect IgG responses against EBOV GP by ELISA.

FIGS. 9A-9B A single immunization with 10¹⁰ vp of rAdC3 Ebola (Zaire)elicits antigen-specific CD4+ and CD8+ T cell responses. Cynomolgusmacaques were vaccinated with rAdC3 or rAd5 encoding EBOV-GP at a doseof 10¹⁰ vp intramuscularly. Blood cells were collected 4 weeks postimmunization to detect cellular immune responses by intracellularcytokine staining after stimulation with EBOV-GP peptides. FIG. 9A shows% cytokine producing CD4+ T cells, FIG. 9B shows % cytokine producingCD8+ T cells.

FIG. 10. A single immunization with 10¹⁰ vp of rAdC3 Ebola (Zaire)protects nonhuman primates against infectious challenge with a lethaldose of EBOV-Zaire. Cynomolgus macaques were vaccinated with rAdC3 orrAd5 encoding EBOV-GP at a dose of 10¹⁰ vp intramuscularly. Subjectswere challenged with 1000 PFU of EBOV-Zaire by the intramuscular routeat 5 weeks post vaccination. *Additional 10 historical controls thatreceived the same vaccine and infectious virus challenge stock haveyielded the same survival result. **More than 50 historical controlsinjected with the same infectious challenge stock have yielded the samesurvival result.

FIGS. 11A-11C rChAd vectors encoding humanized EBOV-GP or non-humanizedEBOV-GP elicited potent immune responses in mice. Five 6-8 weeks femaleBalb/C mice in each group were immunized with rAd EBOV-GP at indicated10⁷ or 10⁸ or 10⁹ viral particles through intramuscular injection. FIG.11A shows CD4 cellular immune responses in PBMC. FIG. 11B shows CD8cellular immune responses in PMBC and FIG. 11C shows humoral responses(IgG) to EBOV-GP. Each were measured at three week post immunization byICS and ELISA, respectively. Zh: humanized EBOV-GP; Z: non-humanizedEBOV-GP; * : p<0.05; **: p<0.01; ***: p<0.001.

FIGS. 12A-12C rChAd prime and boost regimen generated potent immuneresponses in mice. Five 6-8 weeks female Balb/C mice in each group wereimmunized with rAd EBOV-GP at week 0 and boosted at week 3, at 10⁸ or10⁹ viral particles as indicated through intramuscular injection. FIG.12A shows CD4 cellular immune responses in PBMC and FIG. 12B shows CD8cellular immune responses in PBMC. FIG. 12C shows humoral responses(IgG) to EBOV-GP. Each were measured at week 5 by ICS and ELISA,respectively. 5: rAd5; C3: rChAd3; C63: rChAd63; * : p<0.05; **: p<0.01;***: p<0.001.

FIGS. 13A-13C rAd, rMVA and rLCMV vectors used in prime and boostregimen generated potent immune responses in mice. Five 6-8 weeks femaleBalb/C mice in each group were immunized with vectors encoding EBOV-GPat week 0 and boosted at week 4. rAd vectors were dosed at 10⁷ or 10⁸viral particles as indicated, and MVA vectors at 10⁵ pfu, LCMV at 10⁶pfu, through intramuscular injection FIG. 13A shows CD4 cellular immuneresponses in PBMC and FIG. 13B shows CD8 cellular immune responses inPBMC. FIG. 13C shows humoral responses (IgG) to EBOV-GP. Each weremeasured at week 6 by ICS and ELISA, respectively. 5: rAd5; C3: rChAd3;C63: rChAd63; * : p<0.05; **: p<0.01; ***: p<0.001.

DETAILED DESCRIPTION

The present invention also relates to chimpanzee adenovirus vectorswhich include the nucleic acid molecules of the present invention, hostcells which are genetically engineered with the recombinant vectors, theproduction of filovirus polypeptides or fragments thereof by recombinanttechniques and these chimpanzee adenovirus vectors for use in inducingan immune response.

The present invention also relates to pharmaceutical compositions (alsoreferred to as immunogenic compositions) comprising the chimpanzeevectors described above, and a pharmaceutically acceptable diluent,carrier, or excipient carrier as well as to such compositions for use ininducing an immune response. Additionally the compositions may alsocontain an aqueous medium or a water containing suspension, often mixedwith other constituents in order to increase the activity and/or theshelf life. These constituents may be salt, pH buffers, stabilizers(such as skimmed milk or casein hydrolysate), emulsifiers, andpreservatives. An adjuvant may be included in the pharmaceuticalcomposition to augment the immune response to the viral antigenexpressed from the recombinant virus.

Filovirus Antigens

The nucleic acid molecules of the invention may encode structural geneproducts of any filovirus species. There are five species of Ebolaviruses, Zaire (type species, also referred to herein as ZEBOV), Sudan(also referred to herein as SEBOV), Reston, Bundibugyo, and Ivory Coast.There is a single species of Marburg virus (also referred to herein asMARV).

The particular antigen expressed in the vectors of the invention is nota critical aspect of the present invention. The adenoviral vectors ofthe invention can be used to express proteins comprising an antigenicdeterminant of a wide variety of filovirus antigens. In a typicalembodiment, the vectors of the invention include nucleic acid encodingthe transmembrane form of the viral glycoprotein (GP). In otherembodiments, the vectors of the invention may encode the secreted formof the viral glycoprotein (SGP), or the viral nucleoprotein (NP).

One of skill will recognize that the nucleic acid molecules encoding thefilovirus antigenic protein may be modified, e.g., the nucleic acidmolecules set forth herein may be mutated, as long as the modifiedexpressed protein elicits an immune response against a pathogen ordisease. Thus, as used herein, the term “filovirus antigenic protein”refers to a protein that comprises at least one antigenic determinant ofa filovirus protein described above. The term encompasses filovirusantigens (i.e., gene products of a filovirus), as well as recombinantproteins that comprise one or more filovirus antigenic determinants.

In some embodiments, the protein may be mutated so that it is less toxicto cells (see e.g., WO2006/037038). The present invention also includesvaccines comprising a combination of nucleic acid molecules. Forexample, and without limitation, nucleic acid molecules encoding GP, SGPand NP of the Zaire, Sudan and Ivory Coast Ebola strains may be combinedin any combination, in one vaccine composition.

Adenoviral Vectors

As noted above, exposure to certain adenoviruses has resulted in immuneresponses against certain adenoviral serotypes, which can affectefficacy of adenoviral vaccines. The present invention providesadenoviral vectors comprising capsid proteins from chimpanzeeadenoviruses.

Thus, the vectors of the invention comprise a chimpanzee adenoviruscapsid protein (e.g., a fiber, penton or hexon protein). One of skillwill recognize that it is not necessary that an entire chimpanzee capsidprotein be used in the vectors of the invention. Thus, chimeric capsidproteins that include at least a part of a chimpanzee capsid protein canbe used in the vectors of the invention. The vectors of the inventionmay also comprise capsid proteins in which the fiber, penton, and hexonproteins are each derived from a different serotype, so long as at leastone capsid protein is derived from a chimpanzee adenovirus. For example,the fiber protein can be derived from PanAd3, the penton from ChAd3, andthe hexon from ChAd63. In other embodiments, the fiber, penton and hexonproteins can be those provided in SEQ ID NOS:1-9.

In certain embodiments the recombinant adenovirus vector of theinvention is derived mainly or entirely from a chimpanzee adenovirus.Exemplary chimpanzee adenoviruses are known in the art and include, forexample, ChAd3 and ChAd63 (described in WO 2005/071093), and PanAd3,PanAd1, PanAd2, and ChAd83 (described in WO 2010/086189). ChAd 3,ChAd63, and ChAd83 were isolated from the common chimpanzee (Pantroglodytes) and PanAd3, PanAd1, PanAd2 were isolated from the bonobo orpygmy chimpanzee (Pan paniscus).

In some embodiments, the adenovirus is replication deficient, e.g.because it contains a deletion in the E1 region of the genome. Thisallows propagation of such adenoviruses in well known complementing celllines that express the E1 genes, such as for example 293 cells, PER.C6cells, and the like. In certain embodiments, the adenovirus is achimpanzee adenovirus, with a deletion in the E1 and E3 region intowhich an expression cassette encoding the antigen has been cloned. Theconstruction of chimpanzee adenovirus comprising heterologous sequencesencoding antigens is described in WO 2005/071093 and WO 2010/086189.

Typically, a vector of the invention is produced using a nucleic acidcomprising the entire recombinant adenoviral genome (e.g., a plasmid,cosmid, or baculovirus vector). Thus, the invention also providesisolated nucleic acid molecules that encode the adenoviral vectors ofthe invention. The nucleic acid molecules of the invention may be in theform of RNA or in the form of DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.

The adenovirus vectors of the invention are typically replicationdefective. In these embodiments, the virus is renderedreplication-defective by deletion or inactivation of regions critical toreplication of the virus, such as the E1 region. The regions can besubstantially deleted or inactivated by, for example, inserting the geneof interest (usually linked to a promoter). In some embodiments, thevectors of the invention may contain deletions in other regions, such asthe E2, E3 or E4 regions or insertions of heterologous genes linked to apromoter. For E2- and/or E4-mutated adenoviruses, generally E2- and/orE4-complementing cell lines are used to generate recombinantadenoviruses. Mutations in the E3 region of the adenovirus need not becomplemented by the cell line, since E3 is not required for replication.

A packaging cell line is typically used to produce sufficient amount ofadenovirus vectors of the invention. A packaging cell is a cell thatcomprises those genes that have been deleted or inactivated in areplication-defective vector, thus allowing the virus to replicate inthe cell. Suitable cell lines include, for example, PER.C6, 911, 293,and E1 A549.

As noted above, a wide variety of filovirus antigenic proteins can beexpressed in the vectors of the invention. If required, the heterologousgene encoding the filovirus antigenic protein can be codon-optimized toensure proper expression in the treated host (e.g., human). Thus,codon-optimized antigens are also referred to as humanized antigens. Insome embodiments, the viral GP, SGP or NP protein is codon-optimized.For example, codon-optimized antigens include those of SEQ ID NOS: 11,12, and 14. Codon-optimization is a technology widely applied in theart. Typically, the heterologous gene is cloned into the E1 and/or theE3 region of the adenoviral genome.

The heterologous filovirus gene may be under the control of (i.e.,operably linked to) an adenovirus-derived promoter (e.g., the Major LatePromoter) or may be under the control of a heterologous promoter.Examples of suitable heterologous promoters include the CMV promoter andthe RSV promoter. In some embodiments, the promoter is located upstreamof the heterologous gene of interest within an expression cassette.

As noted above, the adenovirus vectors of the invention can comprise awide variety of filovirus antigens known to those of skill in the art.

Immunogenic Compositions

Purified or partially purified adenovirus vectors of the invention maybe formulated as a vaccine (also referred to as an “immunogeniccomposition”) according to methods well known in the art. Suchcompositions may include adjuvants to enhance immune responses. Theoptimal ratios of each component in the formulation may be determined bytechniques well known to those skilled in the art.

The preparation and use of immunogenic compositions are well known tothose of skill in the art. Liquid pharmaceutical compositions generallyinclude a liquid carrier such as water, petroleum, animal or vegetableoils, mineral oil or synthetic oil. Physiological saline solution,dextrose or other saccharide solution or glycols such as ethyleneglycol, propylene glycol or polyethylene glycol may be included.

The compositions are suitable for single administrations or a series ofadministrations. When given as a series, inoculations subsequent to theinitial (priming) administration are given to boost the immune responseand are typically referred to as booster inoculations. The compositionsof the invention can be used as a boosting composition primed by antigenusing any of a variety of different priming compositions, or as thepriming composition. Thus, one aspect of the present invention providesa method of priming and/or boosting an immune response to an antigen inan individual. For example, in some embodiments, a primingadministration of one adenoviral vector of the invention is followed bya booster inoculation of the second adenoviral vector.

The timing of the administration of boosting compositions is well withinthe skill in the art. Boosting compositions are generally administeredweeks or months after administration of the priming composition, forexample, about 2-3 weeks or 4 weeks, or 8 weeks, or 16 weeks, or 20weeks, or 24 weeks, or 28 weeks, or 32 weeks or one to two years.Methods of accelerated vaccination by administering a single dose of arecombinant adenovirus are described for example in U.S. Pat. No.7,635,485.

The compositions of the invention may comprise other filovirus antigensor the priming or boosting inoculations may comprise other antigens. Theother antigens used in combination with the adenovirus vectors of theinvention are not critical to the invention and may be, for example,filovirus antigens, nucleic acids expressing them, virus like particles(VLPs), or prior art viral vectors. Such viral vectors include, forexample, other adenoviral vectors, vaccinia virus vectors, avipoxvectors such as fowlpox or canarypox, herpes virus vectors, vesicularstomatitis virus vectors, or alphavirus vectors. One of skill willrecognize that the immunogenic compositions of the invention maycomprise multiple antigens and vectors.

The antigens in the respective priming and boosting compositions(however many boosting compositions are employed) need not be identical,but should share antigenic determinants.

In some embodiments, heterologous prime-boost approaches can be used,for example, priming with Pan3-EBOV and boosting with ChAd3EBOV, primingwith ChAd3EBOV and boosting with rLCMV (recombinant lymphocyticchoriomeningitis virus), or priming with ChAd63EBOV and boosting withrLCMV. The rLCMV can be constructed as described in Flatz, L. et al.,Nature Medicine, 16:339-345, 2010, except that the sequence encoding theantigenic protein is a sequence encoding a filovirus GP protein of theinvention. In other embodiments, the boost can be rMVA (modifiedvaccinia virus Ankara) encoding an Ebola GP protein. Preparation and useof rMVA vectors is known and described for example in Ourmanov et al. J.Virol. 83:5388-5400, 2009 and Martinon et al. Vaccine 26:532-545, 2008.The vaccines of the invention can be used to generate protection againstall human EBOV threats including Bundibugyo and Ivory Coast in a singlevaccine. Finally, the vaccines against EBOV and MARV may also be mixedinto a single inoculation in order to provide protection against bothfiloviruses simultaneously. In “prime and boost” immunization regimes,the immune response induced by administration of a priming compositionis boosted by administration of a boosting composition. Effectiveboosting can be achieved using replication-defective adenovirus vectors,following priming with any of a variety of different types of primingcompositions, as described for example in U.S. Pat. No. 7,094,598, whichis incorporated herein by reference.

As noted above, the immunogenic compositions of the invention maycomprise adjuvants. Adjuvants suitable for co-administration inaccordance with the present invention should be ones that arepotentially safe, well tolerated and effective in people includingQS-21, Detox-PC, MPL-SE, MoGM-CSF, TiterMax-G, CRL-1005, GERBU,TERamide, PSC97B, Adjumer, PG-026, GSK-I, GcMAF, B-alethine, MPC-026,Adjuvax, CpG ODN, Betafectin, Alum, and MF59.

Other adjuvants that may be administered include lectins, growthfactors, cytokines and lymphokines such as alpha-interferon, gammainterferon, platelet derived growth factor (PDGF), granulocyte-colonystimulating factor (gCSF), granulocyte macrophage colony stimulatingfactor (gMCSF), tumor necrosis factor (TNF), epidermal growth factor(EGF), IL-I, IL-2, IL-4, IL-6, IL-8, IL-10, and IL-12 or encodingnucleic acids therefore.

As noted above, the compositions of the invention may comprise apharmaceutically acceptable excipient, carrier, buffer, stabilizer orother materials well known to those skilled in the art. Such materialsshould be non-toxic and should not interfere with the efficacy of theactive ingredient. The precise nature of the carrier or other materialmay depend on the route of administration, e.g., oral, intravenous,cutaneous or subcutaneous, intramucosal (e.g., gut), intranasal,intramuscular, or intraperitoneal routes. Adiminstration is typicallyintramuscular.

Intramuscular administration of the immunogenic compositions may beachieved by using a needle to inject a suspension of the adenovirusvector. An alternative is the use of a needless injection device toadminister the composition (using, e.g., Biojector™) or a freeze-driedpowder containing the vaccine.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the adenovirus vector will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of skill in the art arewell able to prepare suitable solutions using, for example, isotonicvehicles such as Sodium Chloride Injection, Ringer's Injection, orLactated Ringer's Injection. Preservatives, stabilizers, buffers,antioxidants and/or other additives may be included, as required. Aslow-release formulation may also be employed.

Typically, administration will have a prophylactic aim to generate animmune response against a filovirus antigen before infection ordevelopment of symptoms. Diseases and disorders that may be treated orprevented in accordance with the present invention include those inwhich an immune response may play a protective or therapeutic role. Inother embodiments, the adenovirus vectors can be administered forpost-exposure prophylactics.

The immunogenic compositions containing the adenovirus vectors areadministered to a subject, giving rise to an anti-filovirus immuneresponse in the subject. An amount of a composition sufficient to inducea detectable immune response is defined to be an “immunologicallyeffective dose.” As shown below, the immunogenic compositions of theinvention induce a humoral as well as a cell-mediated immune response.In a typical embodiment the immune response is a protective immuneresponse.

The actual amount administered, and rate and time-course ofadministration, will depend on the nature and severity of what is beingtreated. Prescription of treatment, e.g., decisions on dosage etc., iswithin the responsibility of general practitioners and other medicaldoctors, or in a veterinary context a veterinarian, and typically takesaccount of the disorder to be treated, the condition of the individualpatient, the site of delivery, the method of administration and otherfactors known to practitioners. Examples of the techniques and protocolsmentioned above can be found in Remington's Pharmaceutical Sciences,16th edition, Osol, A. ed., 1980.

Following production of adenovirus vectors and optional formulation ofsuch particles into compositions, the adenovirus vectors may beadministered to an individual, particularly human or other primate.Administration may be to humans, or another mammal, e.g., mouse, rat,hamster, guinea pig, rabbit, sheep, goat, pig, horse, cow, donkey,monkey, dog or cal. Delivery to a non-human mammal need not be for atherapeutic purpose, but may be for use in an experimental context, forinstance in investigation of mechanisms of immune responses to theadenovirus vector.

In one exemplary regimen, the adenovirus vector is administered (e.g.,intramuscularly) in the range of from about 100 μl to about 10 ml ofsaline solution containing concentrations of from about 10⁴ to 10¹²virus particles/ml. Typically, the adenovirus vector is administered inan amount of about 10⁹ to about 10¹² viral particles (vp) to a humansubject during one administration. For example, the adenovirus vectorcan be administered in an amount of about 10⁹, 10¹⁰, 10¹¹, or 10¹² vpper administration. In some embodiments, the dose administered is fromabout 10¹⁰ to about 10¹² vp. An initial vaccination can be followed by aboost as described above. The composition may, if desired, be presentedin a kit, pack or dispenser, which may contain one or more unit dosageforms containing the active ingredient. The kit, for example, maycomprise metal or plastic foil, such as a blister pack. The kit, pack,or dispenser may be accompanied by instructions for administration.

The present invention also provides kits for enhancing the immunity of ahost to a pathogen. These kits may include any one ore more vaccinesaccording to the present invention in combination with a composition fordelivering the vaccine to a host and/or a device, such as a syringe, fordelivering the vaccine to a host.

The vaccine according to the invention is administered as a pre-exposure(or post-exposure) single dose in a manner compatible with the dosageformulation, and in such amount as will be prophylactively effective.Immunity is defined as the induction of a significant level ofprotection after vaccination compared to an unvaccinated human or otherhost.

The vaccine of the present invention, i.e., the recombinant virus, maybe administered to a host, such as a human subject, via anypharmaceutically acceptable routes of administration. The routes ofadministration include, but are not limited to, intramuscular,intratracheal, subcutaneous, intranasal, intradermal, rectal, oral andparental route of administration. Routes of administration may becombined, if desired, or adjusted depending upon the type of thepathogenic virus to be immunized against and the desired body site ofprotection.

Doses or effective amounts of the recombinant virus may depend onfactors such as the condition, the selected viral antigen, the age,weight and health of the host, and may vary among hosts. The appropriatetiter of the recombinant virus of the present invention to beadministered to an individual is the titer that can modulate an immuneresponse against the viral antigen and elicits antibodies against thepathogenic virus from which the antigen is derived. An effective titercan be determined using an assay for determining the activity ofimmunoeffector cells following administration of the vaccine to theindividual or by monitoring the effectiveness of the therapy using wellknown in vivo diagnostic assays.

The chimp Ad vectors of the invention can be used as single inoculationsto provide either immediate (e.g., 2-4 weeks) or long-term (e.g., oneyear) immune protection.

Nucleic Acid Molecules

As indicated herein, nucleic acid molecules of the present invention maybe in the form of RNA or in the form of DNA obtained by cloning orproduced synthetically. The DNA may be double-stranded orsingle-stranded. Single-stranded DNA or RNA may be the coding strand,also known as the sense strand, or it may be the non-coding strand, alsoreferred to as the anti-sense strand. By “isolated” nucleic acidmolecule(s) is intended a nucleic acid molecule, DNA or RNA, which hasbeen removed from its native environment. For example, recombinant DNAmolecules contained in a vector are considered isolated for the purposesof the present invention. Further examples of isolated DNA moleculesinclude recombinant DNA molecules maintained in heterologous host cellsor purified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe DNA molecules of the present invention. Isolated nucleic acidmolecules according to the present invention further include suchmolecules produced synthetically.

Nucleic acid molecules of the present invention include DNA moleculescomprising an open reading frame (ORF) encoding a modified or wild-typefilovirus or adenovirus structural gene product; and DNA molecules whichcomprise a sequence substantially different from those described abovebut which, due to the degeneracy of the genetic code, still encode anORF of a filovirus structural gene product. Of course, the genetic codeis well known in the art.

The present invention is further directed to fragments of the nucleicacid molecules described herein. By a fragment of a nucleic acidmolecule having the nucleotide sequence of an ORF encoding a wild-typefilovirus or adenovirus structural gene product is intended fragments atleast about 15 nt., at least about 20 nt., at least about 30 nt., or atleast about 40 nt. in length. Of course, larger fragments 50, 100, 150,200, 250, 300, 350, 400, 450, or 500 nt. in length are also intendedaccording to the present invention as are fragments corresponding tomost, if not all, of the nucleotide sequence of the ORF encoding awild-type filovirus or adenovirus structural gene product. By a fragmentat least 20 nt. in length, for example, is intended fragments whichinclude 20 or more contiguous bases from the nucleotide sequence of theORF of a wild-type filovirus or adenovirus structural gene product.

In another aspect, the invention provides a nucleic acid moleculecomprising a polynucleotide which hybridizes under stringenthybridization conditions to a portion of the polynucleotide in a nucleicacid molecule of the invention described above. By “stringenthybridization conditions” is intended overnight incubation at 42° C. ina solution comprising: 50% formamide, 5×SSC (750 mM NaCl, 75 mMtrisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmonsperm DNA, followed by washing the filters in 0.1×SSC at about 65° C.

By a polynucleotide which hybridizes to a “portion” of a polynucleotideis intended a polynucleotide (either DNA or RNA) hybridizing to at leastabout 15 nucleotides (nt.), at least about 20 nt., at least about 30nt., or about 30-70 nt. of the reference polynucleotide.

By a portion of a polynucleotide of “at least 20 nt. in length,” forexample, is intended 20 or more contiguous nucleotides from thenucleotide sequence of the reference polynucleotide. Of course, apolynucleotide which hybridizes only to a poly A sequence or acomplementary stretch of T (or U) residues, would not be included in apolynucleotide of the invention used to hybridize to a portion of anucleic acid of the invention, since such a polynucleotide wouldhybridize to any nucleic acid molecule containing a poly A stretch orthe complement thereof (e.g., practically any double-stranded cDNAclone).

As indicated herein, nucleic acid molecules of the present inventionwhich encode a filovirus structural gene product may include, but arenot limited to those encoding the amino acid sequence of the full-lengthpolypeptide, by itself, the coding sequence for the full-lengthpolypeptide and additional sequences, such as those encoding a leader orsecretory sequence, such as a pre-, or pro- or prepro-protein sequence,the coding sequence of the full-length polypeptide, with or without theaforementioned additional coding sequences, together with additional,non-coding sequences, including for example, but not limited to intronsand non-coding 5′ and 3′ sequences, such as the transcribed,non-translated sequences that play a role in transcription, mRNAprocessing, including splicing and polyadenylation signals, for example,ribosome binding and stability of mRNA; and additional coding sequencewhich codes for additional amino acids, such as those which provideadditional functionalities.

The present invention further relates to variants of the nucleic acidmolecules of the present invention, which encode portions, analogs orderivatives of the filovirus or adenovirus structural gene product.Variants may occur naturally, such as a natural allelic variant. By an“allelic variant” is intended one of several alternate forms of a geneoccupying a given locus on a genome of an organism. (Genes II, Lewin,B., ed., John Wiley & Sons, 1985 New York). Non-naturally occurringvariants may be produced using art-known mutagenesis techniques.

Such variants include those produced by nucleotide substitutions,deletions or additions, which may involve one or more nucleotides. Thevariants may be altered in coding regions, non-coding regions, or both.Alterations in the coding regions may produce conservative ornon-conservative amino acid substitutions, deletions or additions. Insome embodiments, the variations are silent substitutions, additions anddeletions, which do not alter the properties and activities of thefilovirus or adenovirus structural gene product or portions thereof. Insome embodiments the variants are conservative substitutions.

Further embodiments of the invention include nucleic acid moleculescomprising a polynucleotide having a nucleotide sequence at least 95%identical, or at least 96%, 97%, 98% or 99% identical to a nucleotidesequence encoding a polypeptide having the amino acid sequence of awild-type filovirus or adenovirus structural gene product or fragmentthereof or a nucleotide sequence complementary thereto.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence encoding a filovirusor adenovirus structural gene product is intended that the nucleotidesequence of the polynucleotide is identical to the reference sequenceexcept that the polynucleotide sequence may include up to five pointmutations per each 100 nucleotides of the reference nucleotide sequenceencoding the virus structural gene product. In other words, to obtain apolynucleotide having a nucleotide sequence at least 95% identical to areference nucleotide sequence, up to 5% of the nucleotides in thereference sequence may be deleted or substituted with anothernucleotide, or a number of nucleotides up to 5% of the total nucleotidesin the reference sequence may be inserted into the reference sequence.These mutations of the reference sequence may occur at the 5′ or 3′terminal positions of the reference nucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongnucleotides in the reference sequence or in one or more contiguousgroups within the reference sequence.

As a practical matter, whether any particular nucleic acid molecule isat least 95%, 96%, 97%, 98% or 99% identical to the reference nucleotidesequence can be determined conventionally using known computer programssuch as the Bestfit program (Wisconsin Sequence Analysis Package,Version 8 for Unix, Genetics Computer Group, University Research Park,575 Science Drive, Madison, Wis. 53711). Bestfit uses the local homologyalgorithm of Smith and Waterman, 1981 Advances in Applied Mathematics2:482-489, to find the best segment of homology between two sequences.When using Bestfit or any other sequence alignment program to determinewhether a particular sequence is, for instance, 95% identical to areference sequence according to the present invention, the parametersare set, of course, such that the percentage of identity is calculatedover the full length of the reference nucleotide sequence and that gapsin homology of up to 5% of the total number of nucleotides in thereference sequence are allowed.

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 95%, 96%, 97%, 98%, or99% identical to a nucleic acid sequence shown herein in the SequenceListing will encode a polypeptide of the invention. In fact, sincedegenerate variants of these nucleotide sequences all encode the samepolypeptide, this will be clear to the skilled artisan even withoutperforming the above described comparison assay. It will be furtherrecognized in the art that, for such nucleic acid molecules that are notdegenerate variants, a reasonable number will also encode a polypeptidehaving the desired activity. This is because the skilled artisan isfully aware of amino acid substitutions that are either less likely ornot likely to significantly affect protein function (e.g., replacing onealiphatic amino acid with a second aliphatic amino acid).

For example, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie, J. U. et al. 1990 Science247:1306-1310, wherein the authors indicate that proteins aresurprisingly tolerant of amino acid substitutions.

Polypeptides and Fragments

The invention further provides filovirus and adenovirus polypeptideshaving the amino acid sequence encoded by an open reading frame (ORF) ofa wild-type or modified filovirus or adenovirus structural gene, or apeptide or polypeptide comprising a portion thereof.

It will be recognized in the art that some amino acid sequences of thefilovirus polypeptides can be varied without significant effect on thestructure or function of the protein. If such differences in sequenceare contemplated, it should be remembered that there will be criticalareas on the protein which determine activity.

Thus, the invention further includes variations of the filovirus oradenovirus polypeptides which show substantial antigenic or otherrelevant biological activity. Such mutants include deletions,insertions, inversions, repeats, and type substitutions. As indicated,guidance concerning which amino acid changes are likely to bephenotypically silent can be found in Bowie, J. U. et al. 1990 Science247:1306-1310.

Thus, the fragment, derivative or analog of the polypeptide of theinvention may be (i) one in which one or more of the amino acid residuesare substituted with a conserved or non-conserved amino acid residue andsuch substituted amino acid residue may or may not be one encoded by thegenetic code, or (ii) one in which one or more of the amino acidresidues include a substituent group, or (iii) one in which additionalamino acids are fused to the mature polypeptide. Such fragments,derivatives and analogs are deemed to be within the scope of thoseskilled in the art from the teachings herein.

Of course, the number of amino acid substitutions a skilled artisanwould make depends on many factors, including those described above.Generally speaking, the number of amino acid substitutions for any givenfilovirus or adenovirus polypeptide will not be more than 50, 40, 30,20, 10, 5 or 3.

Amino acids in the filovirus or adenovirus polypeptides of the presentinvention that are essential for the desired function can be identifiedby methods known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham & Wells 1989 Science244:1081-1085). The latter procedure introduces single alanine mutationsat every residue in the molecule. The resulting mutant molecules arethen tested for biological activity such as changes in immunologicalcharacter.

The polypeptides of the present invention are conveniently provided inan isolated form. By “isolated polypeptide” is intended a polypeptideremoved from its native environment. Thus, a polypeptide produced and/orcontained within a recombinant host cell is considered isolated forpurposes of the present invention. Also intended as an “isolatedpolypeptide” are polypeptides that have been purified, partially orsubstantially, from a recombinant host cell or a native source. Forexample, a recombinantly produced version of the filovirus or adenoviruspolypeptide can be substantially purified by the one-step methoddescribed in Smith and Johnson 1988 Gene 67:31-40.

The polypeptides of the present invention include a polypeptidecomprising a polypeptide having the amino acid sequence of a wild-typefilovirus structural gene product or portion thereof or encoded by anucleic acid sequence shown herein in the Sequence Listing; as well aspolypeptides which are at least 95% identical, or at least 96%, 97%,98%, or 99% identical to those described above.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a reference amino acid sequence of an filovirus oradenovirus polypeptide is intended that the amino acid sequence of thepolypeptide is identical to the reference sequence except that thepolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the reference amino acid of the filovirus oradenovirus polypeptide. In other words, to obtain a polypeptide havingan amino acid sequence at least 95% identical to a reference amino acidsequence, up to 5% of the amino acid residues in the reference sequencemay be deleted or substituted with another amino acid, or a number ofamino acids up to 5% of the total amino acid residues in the referencesequence may be inserted into the reference sequence. These alterationsof the reference sequence may occur at the amino or carboxy terminalpositions of the reference amino acid sequence or anywhere between thoseterminal positions, interspersed either individually among residues inthe reference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular polypeptide is at least95%, 96%, 97%, 98%, or 99% identical to a reference amino acid sequencecan be determined conventionally using known computer programs such asthe Bestfit program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711). When using Bestfit or any other sequencealignment program to determine whether a particular sequence is, forinstance, 95% identical to a reference sequence according to the presentinvention, the parameters are set, of course, such that the percentageof identity is calculated over the full length of the reference aminoacid sequence and that gaps in homology of up to 5% of the total numberof amino acid residues in the reference sequence are allowed.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1

This example shows that humoral and cellular responses generated byChAdC3 Ebola (S/G) and ChAdC3 Ebola (Zaire) were comparable to thosegenerated by rAd 5Ebola (S/G) and rAd5 Ebola (Zaire) respectively.

Immunization of cynomologous macaques with ChAdC3 Ebola (Zaire) producedantigen-specific antibody and Cd4+ and Cd8+ T cell responses. Protectionagainst infection with a lethal dose of EBOV-Zaire was alsodemonstrated, as 4 macaques survived the challenge after immunizationwith ChAdC3 Ebola (Zaire) (see FIGS. 1-7).

Example 2

This example shows that a single immunization with rChAdC3 Ebola (Zaire)elicited humoral and cellular immune responses comparable to thosegenerated by rAd5 Ebola (Zaire).

Immunization of cynomologous macaques with rChAdC3 Ebola (Zaire)produced antigen-specific antibody and Cd4+ and Cd8+ T cell responses.Protection against infection with a lethal dose of EBOV-Zaire was alsodemonstrated, as 4 out of 4 macaques survived the challenge afterimmunization with rChAdC3 Ebola (Zaire) (see FIGS. 8-10).

Example 3

This example shows that a single immunization with adenoviral vectorsencoding humanized Ebola glycoprotein (EBOV-GP) induced strongercellular and humoral responses in mice than adenoviral vectors encodingnon-humanized EBOV-GP.

Groups of female Balb/C mice were immunized with rAd EBOV-GP (Z) at adose of 10⁷, 10⁸, and 10⁹ viral particles via intramuscular injection.Cellular immune responses (Cd4+ and Cd8+ T cell responses) in PBMC andhumoral responses (IgG) to EBOV-GP were measured at three weeks postimmunization by ICS (intracellular cytokine staining) and ELISA,respectively. As shown in FIG. 11, immunization with adenoviral vectorsrChAd3 and rChAd63 encoding EBOV-GP (Zh) codon optimized for expressionin humans produced significantly higher percentages of CD4+ T cells thatexpress cytokines IFN-γ and TNF-α than the same vectors encodingnon-humanized (wild-type) EBOV-GP (Z) (SEQ ID NO:10), and theseresponses were significantly greater than the response due to rAd5 at10⁹ viral particles.

Immunization with rChAd3 encoding humanized EBOV-GP producedsignificantly higher percentages of CD8+ T cells that express cytokinesIFN-γ and TNF-α than the same vector encoding non-humanized EBOV-GP, andthe percentage of cytokine positive cells was comparable, although notsignificantly different, to the percentage of cytokine positive CD8+ Tcells produced by rAd5 at 10⁹ viral particles (see FIG. 11). There wasno significant difference in the CD8+ response produced by rChAd63encoding humanized and non-humanized EBOV-GP.

Immunization with rChAd63 encoding humanized EBOV-GP producedsignificantly higher IgG when compared to the same vector encodingnon-humanized EBOV-GP at 10⁸ viral particles. There was no significantdifference in the IgG response by rChAd3 encoding humanized andnon-humanized EBOV-GP. Further, the IgG response by rChAd3 and rChAd63encoding humanized and non-humanized EBOV-GP was significantly lowerthan response generated by Ad5 (see FIG. 11).

Example 4

This example shows that a prime/boost regimen using adenoviral vectorsencoding EBOV-GP generated potent immune responses in mice.

Groups of female Balb/C mice were immunized with 10⁸ and 10⁹ rAd EBOV-GP(Z) viral particles via intramuscular injection at week 0 and boosted atweek 3. Cellular and humoral immune responses were measured as describedabove at week 5.

As shown in FIG. 12, prime with 10⁹ particles of rChAd3 and boost with10⁹ particles of rChAd63 generated similar CD4+ and CD8+ responses as asingle immunization at 3 weeks with rAd5. Likewise, prime with 10⁹particles of rChAd63 and boost with 10⁹ particles of rChAd3 generated asimilar CD8+ response as a single immunization at 3 weeks with rAd5,whereas this regimen produced a significantly lower CD4+ response.

Prime with 10⁹ particles of rChAd3 and boost with 10⁹ particles ofrChAd63 generated a significantly higher IgG response than a single rAd5immunization at 3 weeks. Similarly, prime with 10⁹ particles of rChAd63and boost with 10⁹ particles of rChAd3 generated a significantly higherIgG response than a single rAd5 immunization at 3 weeks.

As shown in FIG. 13, prime with 10⁸ particles of rChAd63 and boost with10⁸ particles of rChAd3 induced higher CD8+ and IgG responses than primeand boost with rAd5. The LCMV and MVA vectors were prepared as describedabove.

In summary, the above examples demonstrate that rChAd3 consistentlygenerated comparable immune responses as rAd5 for single administration.Further, prime and boost with rChAd3/rChAd63, ChAd63/ChAd3, andChAd3/LCMV are useful candidates for a combination regimen.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

What is claimed is:
 1. An isolated nucleic acid molecule encoding arecombinant adenovirus vector comprising nucleic acid encoding afilovirus antigenic protein, wherein the adenovirus vector comprises achimpanzee adenovirus capsid protein.
 2. The isolated nucleic acidmolecule of claim 1, comprising an expression cassette comprising a CMVpromoter operably linked to a polynucleotide sequence encoding thefilovirus antigenic protein.
 3. The isolated nucleic acid molecule ofclaim 1, which comprises a sequence at least 95% identical to a sequenceselected from the group consisting of SEQ ID NOs: 1-9.